Human cells are constantly exposed to a variety of hormones, growth factors and other agents that affect cell growth. To integrate and interpret these external stimuli, complex signaling networks have evolved, which allow different types of cells to respond appropriately to their environment. In all mammals the reversible phosphorylation of proteins regulates many intracellular signaling networks that control cell growth, differentiation, senescence and programmed cell death (apoptosis). Protein phosphorylation occurs principally on serine, threonine and tyrosine residues, and the phosphorylation reaction is catalyzed by a large family of protein kinases. To date many compounds that function as potent and highly selective inhibitors of "key" protein kinases have been identified, and these inhibitors have proven to be powerful tools to probe the biology and pathology associated with the actions of protein kinases. In contrast, much less is known about the biology of protein phosphates. Notably the biological roles and pathology associated with phosphates belonging to the PP2C subfamily are poorly understood. To a large extent, this is due to the lack of probes. Unlike dual specificity and tyrosine phosphates, where the Cys-based catalytic mechanism allows for the development of substrate trapping mutants, the metal-based catalytic mechanism of PP2C-enzymes cannot be modified to produce substrate-trapping mutants. In addition, natural compounds that potently act on PPP-family phosphates (i.e. okadaic acid and calyculin A), do not affect PP2C activity. Therefore, tools to study PP2C-family phosphates are particularly desired by the research community. Having developed methods to produce a large amount of catalytically active PP2C4 (a known oncogene) and a fluorescent assay to reliably measure PP2C4 activity, the objective of this application is to develop a high throughput ready assay that can be used to identify specific, or highly selective, inhibitors for this biologically important phosphatase. The compounds produced from this effort will serve as powerful small molecule probes that will greatly aid efforts to elucidate the roles of PP2C4 in normal biology and human disease. They may also serve as lead compounds for the development of new drugs for medical management of human cancer. PUBLIC HEALTH RELEVANCE: Serine/threonine protein phosphatase 2C delta (PP2C4) has emerged as a regulator of p53 signaling networks and appears to act as an oncogene. Having developed methods to produce large amounts of (PP2C4) and an assay to measure (PP2C4) activity, we proposal is to identify a specific or highly selective inhibitor by developing methods that will enable a large scale screen in conjunction with the Molecular Libraries Probe Production Centers Network (MLPCN). The compounds produced from this effort will serve as powerful tool to probe the biology and pathology associated with (PP2C4).